The large increase in the use of sedatives including barbituric acids by the general population has brought with it a substantial need to improve analytical techniques for the determination of such materials in biological fluids. In many instances, medical treatment centers are faced with the need of determining the identity of a sedative taken by a patient who, being in a comatose condition, is unable to supply such information to the treating physician. There has also been much publicity in recent years concerning drug abuse, particularly abuse of sedatives such as barbituric acid derivatives.
At present, procedures for the identification of barbituric acid derivatives involve extraction and thin layer chromatographic methods. These techniques have the disadvantage of being relatively time consuming, laborious and lacking great sensitivity. A more rapid and highly sensitive assay for the presence of barbituric acid derivatives in biological fluids would thus represent an extremely important advance in the art.
It has been known in the art for some time that various small molecules (haptens), which by themselves are wholly devoid of antigenicity, can modify the antigenic properties of a protein when a small molecule is combined with the protein through stable covalent linkages. In U.S. Pat. No. 2,372,066, patented Mar. 20, 1945, it is disclosed that antigens may be prepared by combining histamine or histamine-like compounds by linking the imidazole ring to a desired protein through a radical containing a group capable of coupling with the protein. These antigens are used either by direct injection into a subject whereby resistance, refractoriness or active immunity is developed in said subject or for injecting into host animals from which antibodies specific to the hapten moiety, e.g. the histamine or histamine-like substance are developed.
A similar contemporary disclosure was made by Landsteiner in the "Specificity of Serological Reactions", Harvard University Press, Cambridge, Massachusetts (1945) wherein p-amino benzene arsonic acid was coupled to a protein via its diazonium salt to form a chemically simple, well-defined compound which was antigenic and stimulated the production of antibodies. Furthermore, the antibodies to this immunogen (conjugated protein) can combine with the small molecule, e.g. the arsonic acid which is unattached to any protein. This antibody is quite specific in activity. For example, if an isomer of arsonilic acid, in which the --AsO.sub.3 H group is in the meta position relative to the amino group, is utilized, it will not combine with the antibody formed against the protein-arsonilic acid complex in which the --AsO.sub.3 H group is para to the amino group.
It should be mentioned that it is not yet possible, in the present state of the art, to predict or determine what properties are required to enable a molecule to act as an antigen. At one time, molecular weight and the possession of an aromatic group were thought to be the deciding factors. With time, the critical molecular weight required for antigenicity has been remarkably reduced. It is still believed, however, that the molecular weight will, to some extent, determine the antigenic capabilities of a molecule. Other factors such as molecular shape and chemical reactivity must also play a role in the antigenic properties and thus render prediction of such properties exceedingly more difficult.